The family Papovaviridae constitutes a group of DNA viruses that induce both lytic infections and either benign or malignant tumors. Structurally, all are naked icosahedral virions with 72 capsomeres and contain double-stranded circular DNA. Viruses included in the family are: (1) human and animal papillomaviruses, (2) mouse polyomavirus, (3) simian vacuolating virus, and (4) human viruses BK and JC.
Human papillomaviruses (HPV) infect cutaneous, genital, oral, and respiratory epithelia in a tissue-specific manner. Infection with HPV has been associated closely with the development of both benign lesions and malignancies (Reichman et al., Papillomaviruses, 1990, pp. 1191-1200; and Mandell et al., Principles and Practice of Infectious Diseases, 3rd Edition, Churchill Livingstone, New York, N.Y.). For example, HPV type 1 (HPV-1) is present in plantar warts, HPV types 6 or 11 (HPV-6 or HPV-11) in condylomata acuminata (anogenital warts), while HPV types 16 or 18 (HPV-16 or HPV-18) are common in premalignant and malignant lesions of the cervical squamous epithelium (See Crum et al., "Human papillomavirus infection and cervical neoplasia: New perspectives," Int. J. Gynecol. Pathol. 3:376-388 (1984); zur Hausen, Genital Papillomavirus Infections, 1985, pp. 83-90; Rigby et al., Viruses and Cancer, Cambridge University Press, Cambridge, UK; and Koutsky et al., "Epidemiology of genital human papillomavirus infection," Epidemiol. Rev. 10:122-163 (1988)).
However, difficulties in propagating HPV in vitro has led to the development of alternative approaches to antigen production for immunologic studies. For example, Bonnez et al., "The Pstl-XhoII restriction fragment of the HPV-6b L1 ORF lacks immunological specificity as determined by sera from HPV 6 condyloma acuminatum patients and controls," UCLA Symp. Mol. Cell. Biol., New Series, 124:77-80 (1990); Jenison et al., "Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins," J. Virol. 62:2115-2123 (1988); Li et al., "Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera," J. Virol. 61:2684-2690 (1987); Steele et al., "Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1," Virology 174:388-398 (1990); and Strike et al., "Expression in Escherichia coli of seven DNA segments comprising the complete L1 and L2 open reading frames of human papillomavirus type 6b and the location of the `common antigen`," J. Gen. Virol. 70:543-555 (1989), have expressed recombinant capsid protein coding sequences in prokaryotic systems, and used them in Western blot analyses of sera obtained from individuals with HPV infection of the genital tract. Results from these studies have suggested that antibodies to denatured, i.e. linear, epitopes of HPV capsid proteins can be detected in the sera of some infected individuals.
Whole virus particles have also been used to detect antibodies in human sera, including antibodies directed against conformational epitopes. These studies have been difficult to conduct because most naturally occurring HPV-induced lesions produce few particles. Whole virus particles can be obtained, however, in amounts sufficient to conduct immunologic assays from HPV type 1-induced plantar warts (Kienzler et al., "Humoral and cell-mediated immunity to human papillomavirus type 1 (HPV-1) in human warts," Br. J. Dermatol. 108:65-672 (1983); "Pfister et al., Seroepidemiological studies of human papilloma virus (HPV-1) infections," Int. J. Cancer 21:161-165 (1978); and Steele et al., "Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1," Virology 174:388-398 (1992)) and experimentally-induced HPV-11 athymic mouse xenographs (Kreider et al., "Laboratory production in vivo of infectious human papillomavirus type 11," J. Virol. 61:590-593 (1991); and Kreider et al., "Morphological transformation in vivo of human uterine cervix with papillomavirus from condylomata acuminata," Nature 317:639-641 (1985)). More particularly, U.S. Pat. No. 5,071,757 to Kreider et al., discloses a method of propagating infectious HPV-11 virions in the laboratory using an athymic mouse xenograph model system. Although this system is capable of producing quantities of infectious virus that could be used for the development of a serologic test for genital HPV infection, this system is very expensive and cumbersome. Furthermore, only one genital HPV type has so far been propagated in this system, thus, limiting its usefulness. In addition, the infectious virus produced using this system represents a biohazard and, therefore, would be difficult to use in a vaccine formulation.
Zhou et al., in "Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion-like particles", Virology 185:251-257 (1992), have reported the formation of HPV-16 virus-like particles in CV-1 cell nuclei following infection with a vaccinia virus HPV-16 L1/L2 double recombinant expression vector. However, the authors were not able to produce VLPs with a vector expressing L1 alone. Furthermore, the VLPs produced lacked a well-defined symmetry, and were more variable in size and smaller, only about 35-40 nm in diameter, than either HPV virions (55 nm) or the VLPs of the present invention (baculovirus produced HPV-11 VLPs, about 50 nm in diameter).
U.S. Pat. No. 5,045,447, to Minson, discloses a method of screening hybridoma culture supernatants for monoclonal antibodies with desired specificities. Minson's method is exemplified by the production of antibodies to the L1 protein of human papillomavirus type 16 (HPV-16) using this protein as the target antigen in mice. However, Minson fails to disclose the expression of the L1 protein or production of HPV virus-like particles (VLPs).
U.S. Pat. No. 4,777,239, to Schoolnik et al., discloses short peptide sequences derived from several of the papillomavirus early region open reading frames which elicit type-specific antibodies to papillomavirus. However, the inventors fail to disclose any sequences directed to the major late open reading frame, L1.
U.S. Pat. No. 5,057,411 to Lancaster et al., discloses a polynucleotide sequence of about 30 nucleotides of the papillomavirus L1 capsid protein open reading frame that the inventors contend encode a papillomavirus type-specific epitope. However, the inventors do not disclose infected animals that produced antibodies which recognize this sequence. Instead, they synthesized a bovine papillomavirus type 1 (BPV-1) version of the sequence (a 10 amino acid peptide, or decapeptide), then immunized rabbits and tested the antiserum's ability to react with either BPV-1 or BPV-2 induced fibropapilloma tissue. The peptide antiserum only reacted with BPV-1 and not BPV-2 tissue. The inventors then concluded that the peptide contained an antigenic determinant that was type-specific, and therefore, all papillomavirus L1 coding sequences contain a type-specific epitope at this locus. This is theoretical speculation on the part of the inventors, who give no supporting data for this hypothesis. In addition, the amino acid sequences disclosed (10 amino acids) are generally thought not to be capable of adopting higher order antigenic structures, i.e., conformational epitopes that possess a three-dimensional structure such as those produced by the method described herein.
Another problem associated with papillomavirus infections is the need for alternative therapeutic and prophylactic modalities. One such modality which has received little recent study, would be papillomavirus vaccines. In 1944, Biberstein treated condyloma acuminatum patients with an autogenous vaccine derived from the patients' warts (Biberstein, "Immunization therapy of warts," Arch. Dermatol Syphilol. 50:12-22 (1944)). Thereafter, Powell et al., developed the technique typically used today for preparing autogenous wart vaccines for the treatment of condyloma acuminatum (Powell et al., "Treatment of condylomata acuminata by autogenous vaccine," South Med. J. 63:202-205 (1970)). Only one double-blind, placebo-controlled study has attempted to evaluate the efficacy of the autogenous vaccine (Malison et al., "Autogenous vaccine therapy for condyloma acuminatum: A double-blind controlled study," Br. J. Vener. Dis. 58:62-65 (1982)). The authors concluded that autogenous vaccination was not effective in the treatment of condylomata acuminata, although this interpretation may be erroneous. The small number of patients studied precluded drawing valid negative conclusions. In any event, autogenous vaccines, as presently described, have several disadvantages. First, the patient needs to have relatively large warts (2 g to 5 g) in order to prepare the vaccine. Secondly, the practitioner needs access to laboratory equipment and expertise each time a new patient is to be treated. Thus, vaccine preparation is very expensive, tedious, and in cases involving relatively small lesion mass, not possible.
Unfortunately, traditional methods of virus propagation have not yet been adapted to the study of papillomaviruses, and the alternative methods previously described fail to produce infectious virions in any significant amounts for immunologic studies. Also, in vivo propagation of HPV-11 in the athymic mouse system is not very practical because it is expensive, labor intensive and currently limited to HPV-11. Consequently, an alternative method of producing epitopes of HPV capsid for use in immunologic studies and vaccine production is needed.